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Abstract
1999;96:1692-1697. 1
2017;158:1120-1129. 1
Abstract
9 10 Obesity is characterized by the excessive accumulation of adipose tissue, and it is a serious 11 global health issue. Understanding the pathology of obesity is crucial for developing effective 12 interventions. In this study, we investigated the role of muscarinic acetylcholine receptor 13 M4 (mAChR-M4) in the regulation of obesity in Chrm4-knockout (M4-KO) mice. Male M414 KO mice showed higher weight gain and accumulation of white adipose tissue (WAT) with 15 advancing age when compared to the wild-type mice. The M4-KO mice also showed 16 increased leptin expression at both the transcription and translation levels. RNA sequencing 17 AC CE PT ED M AN US CR IP T Doloaded rom http/academ ic.p.com /endo/advance-arti1210/endocr/bqaf064/8105668 by gest on 07 April 2025
10 Obesity is characterized by the excessive accumulation of adipose tissue, and it is a serious 11 global health issue. Understanding the pathology of obesity is crucial for developing effective 12 interventions. In this study, we investigated the role of muscarinic acetylcholine receptor 13 M4 (mAChR-M4) in the regulation of obesity in Chrm4-knockout (M4-KO) mice. Male M4-14 KO mice showed higher weight gain and accumulation of white adipose tissue (WAT) with 15 advancing age when compared to the wild-type mice. The M4-KO mice also showed 16 increased leptin expression at both the transcription and translation levels. RNA sequencing 17 AC CE PT ED M AN /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 and quantitative reverse transcription polymerase chain reaction analyses of subcutaneous 1 adipose tissues revealed that the expression of WAT marker genes was significantly 2 enhanced in the M4-KO mice. In contrast, the expression levels of brown adipose 3 tissue/beige adipose tissue markers were strongly decreased in the M4-KO mice. To identify 4 the Chrm4-expressing cell types, we generated Chrm4-mScarlet reporter mice and 5 examined the localization of the mScarlet fluorescent signals in subcutaneous tissues. 6 Fluorescent signals were prominently detected in WAT and mesenchymal stem cells. 7 Additionally, we also found that choline acetyltransferase was expressed in macrophages, 8 suggesting their involvement in acetylcholine (ACh) secretion. Corroborating this notion, we 9 were able to quantitatively measure the ACh in subcutaneous tissues by liquid 10 chromatography tandem mass spectrometry. Collectively, our findings suggest that 11 endogenous ACh released from macrophages maintains the homeostasis of adipose cell 12 growth and differentiation via mAChR-M4 in male mice. This study provides new insights 13 into the molecular mechanisms underlying obesity and potential targets for therapeutic 14 interventions. 15 16 Abbreviations: KO, knock-out; KI, knock-in; WAT, white adipose tissue; WT, wild type; BAT, 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 brown adipose tissue; AD-MSCs, adipose-derived mesenchymal stem cells; ACh, 1 acetylcholine; mAChR, muscarinic acetylcholine receptor; qRT-PCR, quantitative reverse 2 transcription polymerase chain reaction; HFD, high-fat diet; GLP-1, glucagon-like peptide-3 1; NPY, neuropeptide Y; ChAT, choline acetyltransferase; GAPDH, glyceraldehyde-3-4 phosphate dehydrogenase; NPGL, neurosecretory protein GL; NPGM, neurosecretory 5 protein GM; MCH, melanin-concentrating hormone; Chrm4, gene encoding muscarinic 6 acetylcholine receptor M4; mAChR-M4, muscarinic acetylcholine receptor M4; WAT, white 7 adipose tissue; LC-MS/MS, liquid chromatography tandem mass spectrometry 8 9 Introduction 10 11 Aging is an extremely complex multifactorial process characterized by functional decline 12 over time. Obesity and metabolic disorders that accompany aging are serious global health 13 problems that involve both physical and emotional signs and symptoms. As many factors 14 contribute to the age-related increase in adiposity, the accumulation of more evidence is 15 needed to better understand the biological and pathological mechanisms underlying the 16 excessive gain of body fat. White adipose tissue (WAT) functions as an energy reservoir and 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 as an endocrine organ. Some cytokines are overexpressed in the WAT of obese individuals 1 and can lead to diseases related to chronic inflammation (1). It has been shown that the age-2 related decline in metabolism is partly due to reduced muscle mass (2). Additionally, the 3 age-related decline in metabolic thermogenesis in brown adipose tissue (BAT) has been 4 strongly linked to the accumulation of body fat in mid-aged individuals (3). The discovery of 5 effective and safe pharmacological options for preventing and treating obesity is expected to 6 provide new avenues for improving metabolic health. However, better understanding of the 7 pathways and mechanisms that underlie weight gain with advancing age is needed. 8 Muscarinic acetylcholine receptors (mAChRs) are a family of G-protein coupled 9 receptors that are involved in muscarinic signaling. There are five subtypes of mAChRs (M1 10 to M5) and they are expressed in both the central and peripheral nervous systems. They are 11 also expressed in non-neuronal tissues, where they mainly play pivotal roles in the digestive 12 system (4-7). At the molecular level, the M4 subtype of mAChR (mAChR-M4) couples to G 13 proteins of the Gi/Go family. mAChR-M4 is expressed abundantly in the striatum, a region 14 known to be critically involved in extrapyramidal motor control, and mAChR-M4 is known 15 to exert inhibitory control on D1 dopamine receptor-mediated locomotor stimulation (8). In 16 non-neuronal tissues, mAChR-M4 is expressed in both keratinocytes and melanocytes, and 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 has been shown to control murine hair follicle cycling and pigmentation (9-11). However, the 1 role of ACh signaling via mAChR-M4 in obesity with advancing age remains obscure. 2 In the present study, we employed a loss-of-function mouse model to investigate the 3 role of mAChR-M4 in body weight homeostasis in adipose tissues. We found a relationship 4 between neurosecretory protein GL (NPGL) (12) and mAChR-M4 in the central nervous 5 system. Our results revealed a novel function of mAChR-M4 in controlling the body weight 6 and homeostasis of adipose cell growth and differentiation. 7 8 Materials and Methods 9 10 Animal Experiments 11 12 Animals 13 14 The global M1-M5-/- (M1- to M5-KO) mice and wild-type (WT) mice on the C57BL/6 15 background have been described previously (8,13−16). Male mice were singly housed or 16 housed in a group of three at all times in a temperature- and humidity-controlled 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 environment (22C ± 2C, 33% to 35% humidity) on a 12-h light-dark cycle. Mice were 1 maintained on a standard chow diet (CE-2; CLEA Japan, Tokyo, Japan). For the high-fat 2 diet (HFD)-induced obesity experiment, age-matched WT and M4-KO mice were single-3 housed and maintained on a HFD in which fat accounted for 60% of the calories (HFD-60; 4 OrientalBio, Tokyo, Japan) for 12 weeks. Body weight and food intake were measured 5 weekly for 12 weeks. The weekly food intake was determined by subtracting the mass of the 6 food remaining from that supplied for the singly housed mice at the beginning. All animal 7 experiments were approved by the Suntory animal ethics committee (APRV000024 and 8 APRV000561) and were performed according to the institutional guidelines. Mice were 9 euthanized by CO2 asphyxiation. 10 11 Generation of Chrm4-mScarlet knock-in and IRES-mScarlet reporter mice 12 13 To generate a Chrm4-mScarlet knock-in (M4-KI) mouse line, we employed the 14 CRISPR/Cas9-mediated knock-in (KI) transgenesis approach using mouse extended 15 pluripotent stem cells (EPSCs), which have been demonstrated to be advantageous in 16 rapidly generating fully EPSC-derived chimeric mice in the F0 generation. To enable 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 CRISPR/Cas9-mediated insertion through homology-directed repair, we generated an all-in-1 one Cas9 plasmid (#87108; Addgene, Tokyo, Japan) harboring the SpCas9 gene, a guide RNA 2 sequence targeting the Chrm4 locus, and a puromycin resistance cassette for drug selection. 3 A donor template containing homology arms covering the approximately 800 bp upstream 4 and downstream of the Chrm4 locus was constructed by assembling a DNA fragment 5 encoding the mScarlet-I reporter gene (#85044; Addgene) with DNA sequences containing 6 the woodchuk hepatitis virus posttranscriptional regulatory element (WPRE) and SV40 7 polyadenylation signal using an NEBuilder kit (#E2621; New England Biolabs, Tokyo, 8 Japan). 9 To obtain KI EPSC clones in which an mScarlet-I reporter cassette was inserted into 10 the translation initiation site of the Chrm4 locus, we co-transfected EPSCs with the all-in-11 one Cas9 plasmid along with the donor template DNA using the TransIT-2020 transfection 12 reagent (#MIR5404; Mirus Bio, Madison, USA). After puromycin selection to enrich the 13 transfected cells, the surviving EPSCs were further cultured on mitomycin-treated feeder 14 cells in a 10-cm dish supplemented with N2B27 medium containing 103 U/mL of leukemia 15 inhibitory factor, 3 μM CHIR99021 (4423; Tocris, Bristol, United Kingdom), 2 μM (S) -(+)-16 dimethindene maleate (1425; Tocris), and 2 μM minocycline hydrochloride (M9511; Sigma, 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Kawasaki, Japan). The formed EPSC colonies were picked and screened by reverse 1 transcription polymerase chain reaction (RT-PCR) genotyping to select those with correctly 2 integrated sequences in the desired genomic locus. The EPSCs harboring the mScarlet 3 reporter were microinjected into 8-cell stage embryos (ICR; SLC, Hamamatsu, Japan). Then, 4 the injected embryos were transferred to the oviducts of E0.5 pseudo-pregnant females. The 5 resulting chimeric mice that were born were used for this study. 6 Chrm4-IRES-mScarlet KI mice were generated in a similar manner as the M4-KI 7 mice using the CRISPR/Cas9-mediated KI transgenesis approach in EPSCs. The IRES-8 mScarlet-pA cassette was inserted downstream of the stop codon to maintain the 9 endogenous expression of Chrm4. As expected, the homozygous Chrm4-IRES-mScarlet mice 10 were healthy and fertile. 11 12 Blood analysis 13 14 Random blood glucose was measured with a glucometer (Glutest Neo Alpha Sensor; Sanwa 15 Kagaku Kenkyusho, Kyoto, Japan) using blood collected from the tail vein. For serum 16 collection, mice were euthanized by CO2 asphyxiation, and blood was collected from the 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 heart. The whole blood was kept at room temperature until it clotted, then it was centrifuged 1 at 2000 × g at room temperature for 10 min to separate the serum. The serum concentrations 2 of insulin and leptin were measured with an Ultra Sensitive Mouse Insulin ELISA kit 3 (M1104; MORINAGA, Yokohama, Japan) (Table 1) and Mouse and Rat Leptin ELISA kit 4 (RD291001200R; BioVendor, Brno, Czech Republic) (Table 1), respectively, according to the 5 manufacturer’s protocols. Serum samples were stored at -80°C until assayed. For measuring 6 the glucagon-like peptide-1 (GLP-1) levels, blood samples were collected into lithium 7 heparin anticoagulant tubes, and plasma was prepared by centrifugation of the samples at 8 2000 × g for 10 min at 4°C. The concentration of GLP-1 in the plasma was measured with a 9 GLP-1 (9-36/37) Assay Kit (27788; IBL, Gunma, Japan) (Table 1) according to the 10 manufacturer’s protocol. Plasma samples were stored at -80°C until assayed. 11 12 Quantitative analysis of NPY 13 14 Neuropeptide Y (NPY) crude peptides were extracted from WT and M4-KO mouse brains as 15 previously described (17). After solid-phase extraction, the NPY peptides were applied to 16 gel-filtration chromatography as previously described (17). 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Quantitative data on NPY were acquired using Nexera Micros HPLC and LC-MS 1 8060 triple-quadrupole MS/MS instruments (Shimadzu, Kyoto, Japan) in which a Shim-2 pack MC C18 column (0.3 × 50 mm, 1.9 μm; Shimadzu) was used as an analytical column. 3 The trap column was an InertSustainSwift C18 cartridge (2.1 × 10 mm, 5 μm; GL Sciences, 4 Tokyo, Japan). The Nexera Micros HPLC system has a MicrosLC gradient solvent elution 5 system for the analytical column and an isocratic elution HPLC system for the trap column. 6 Solvents A (formic acid aqueous solution) and B (acetonitrile with 0.1% formic acid) were 7 used in the MicrosLC. The flow rate was 14 μL/min, and the gradient program from solvent 8 A to B was as follows: the solvent B content was increased from 22% B to 38% B over 21 min. 9 The transition of the triple quadrupole MS/MS was m/z 712.80 to m/z 751.10 for NPY. The 10 retention time was about 15.8 min for NPY. The crude extract (1 mL) derived from gel-11 filtration chromatography was divided into five aliquots (200 µL each). Each aliquot was 12 lyophilized using a freeze-dryer (FDU-2110; EYELA, Osaka, Japan). After the lyophilized 13 samples were dissolved in 67 µL of water, an aliquot of 14 µL was injected into the microLC-14 MS/MS apparatus for quantitative analysis. 15 16 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Gene expression analysis ( qRT-PCR) 1 2 Brain tissue, subcutaneous tissue, liver tissue, and interscapular BAT were dissected from 3 WT and M4-KO mice. They were immediately frozen with liquid nitrogen and stored at -4 80°C. For the experiments, tissues were homogenized in Trizol reagent (Sepasol-RNA I 5 Super G; Nacalai Tesque, Kyoto, Japan), then incubated for 5 min at room temperature. 6 Chloroform (200 µL; Nacalai Tesque) was added, and the tubes were shaken vigorously, then 7 incubated for 3 min at room temperature. The samples were then centrifuged at 18,000 × g 8 for 15 min at 4°C. The aqueous phase was mixed 1:1 with isopropanol (Nacalai Tesque) , then 9 incubated for 10 min on ice. Subsequently, the samples were centrifuged at 18,000 × g for 10 15 min at 4°C. To remove genomic DNA, the pellets were treated with DNase I (TURBO 11 DNase; Ambion, Austin, TX, USA) for 15 min at 37°C. To stop the reaction and precipitate 12 the RNA, 30 µL of nuclease-free water and 30 µL of LiCl precipitation solution were added. 13 The samples were mixed thoroughly, then chilled for 30 min at -20°C. The RNA solutions 14 were centrifuged at 20,000 × g for 15 min at 4°C to pellet the RNA. The RNA content was 15 measured using NanoDrop (DS-11; Thermo Fisher, Waltham, MA, USA). Subsequently, the 16 RNA (1-µg) was used as a template for cDNA synthesis. Reverse transcription was 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 performed with SuperScript II and oligo-dT primers (3’ RACE System; Invitrogen, Carlsbad, 1 CA, USA) according to the manufacturer’s protocol. 2 Specific gene expression levels were analyzed by quantitative RT-PCR (qRT-PCR). 3 The qRT-PCR for specific genes was performed in triplicate using SYBR Green Master Mix 4 (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. qRT-PCR was 5 carried out on a CFX96 Real-Time System (Bio-Rad) with the following conditions: 6 polymerase activation and DNA denaturation for 30 s at 95°C, followed by 45 cycles at 95°C 7 for 10 s and 55°C for 30 s, then 65°C for 5 s, and finally 95°C for 50 s for the melt-curve 8 analysis. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was amplified as 9 an internal control. All primers for qRT-PCR are shown in Supplementary Table S1 (18). As 10 the region between the middle of the second transmembrane domain and the N terminus of 11 the third intracellular loop of the mAChR-M4-coding sequence was disrupted (8), we 12 designed the primers to be the site of the disruption. For the relative quantification of the 13 gene expression level, the ΔΔCT method was used. 14 15 16 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 RNA sequencing 1 2 Total RNA was extracted from the subcutaneous adipose tissues of WT and M4-KO mice and 3 purified and depleted genomic DNA as described above. Three individual samples were 4 collected and used for RNA sequencing (RNA-seq). An aliquot (500 ng) of quality-confirmed 5 RNA was used for library construction, and was sequenced by Novagene (Beijing, China) 6 using the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). The resulting 7 fastq files were analyzed and deposited into the National Center for Biotechnology 8 Information (NCBI) database (Accession No. SRR29366322-SRR29366333). The reads in 9 these fastq files were mapped to mouse mm10 genome using Hisat2 (v2.2.1) and the 10 resulting gene expression levels are estimated as exported values of transcripts per million 11 (TPM) using cufflinks (v2.2.1). The RNA-seq data were confirmed by qRT-PCR as described 12 above. 13 14 Bioinformatics analysis 15 16 Differential expression and gene ontology (GO) enrichment analyses were conducted using 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 iDEP.951 (http://bioinformatics.sdstate.edu/idep95/). Briefly, the normalized TPM 1 expression values were imported into iDEP.951 and subjected to log transformation, then 2 the transcripts with a low abundance (<1 TPM) were removed from the analysis. 3 Differentially expressed genes (DEGs; genes with a 1.5-fold change and a false discovery 4 rate (FDR)-adjusted p < 0.05) were then subjected to GO enrichment analysis using 5 iDEP.951. 6 7 Immunofluorescence staining 8 9 For immunofluorescence staining experiments, subcutaneous adipose tissue was dissected 10 from WT, M4-KI, and M4-reporter mice. The tissues were fixed with 4% paraformaldehyde 11 (Nacalai Tesque) for 3 h at 4°C, followed by washing with phosphate-buffered saline (PBS; 12 pH 7.4) and an overnight incubation in 15% (w/v) sucrose. Tissue samples were prepared for 13 cryosectioning as described previously (19). The samples were incubated in a 15% sucrose-14 7.5% gelatin solution for 1 h at 37°C. Then, the samples were placed in Tissue-Tek 15 Cryomolds (Sakura Finetek, Torrance, CA, USA) filled with a warm sucrose-gelatin solution. 16 The Cryomolds were incubated for 20 min at 4°C to allow the sucrose-gelatin solution to 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 solidify. The obtained blocks were trimmed and stored at 4°C. The blocks were subsequently 1 embedded in Tissue-Tek OCT compound using new Tissue-Tek Cryomolds and stored at -2 80°C before sectioning (10 µm) on a cryostat (CryoStar NX70; Thermo Scientific, Waltham, 3 MA, USA). Before immunofluorescence staining, sections were dried for 1 h at 37°C. 4 Subsequently, they were washed twice with PBS and PBS containing 0.2% Triton X-100 5 (PBST; Nacalai Tesque) for 2 min and 5 min in series, respectively. The sections were then 6 treated with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) dissolved 7 in PBST for 60 min at room temperature. The primary and secondary antibodies used for 8 immunofluorescence (Table 1) were diluted with 1% BSA/PBST. 9 For the double immunofluorescence staining of mScarlet and perilipin-1, sections 10 were incubated overnight with a combination of rat anti-red fluorescent protein (REF) 11 antibody (1:250) and rabbit anti-perilipin-1 antibody (1:400) at 4°C. After washing twice 12 with PBS for 2 min, the sections were incubated for 1 h with a combination of eFluor 570 13 mouse anti-rat IgG2a (1:1000) and Alexa Fluor Plus 488 goat anti-rabbit IgG (1:1000) at 14 room temperature. 15 For the triple immunofluorescence staining of mScarlet, CD34, and CD105, sections 16 were first incubated overnight with rat anti-REF antibody (1:250) at 4°C. After washing 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 twice with PBS for 2 min, the sections were incubated for 1 h with eFluor 570 mouse anti-1 rat IgG2a (1:1000) at room temperature. Next, the sections were incubated for 1 h with a 2 combination of rat anti-CD34 monoclonal antibody conjugated with fluorescein 3 isothiocyanate (FITC; 1:100) and rat anti-CD105 monoclonal antibody conjugated with 4 Alexa Fluor 647 (1:100) at room temperature. 5 For the double immunofluorescence staining of F4/80 and choline acetyltransferase 6 (ChAT), sections were incubated overnight with a combination of rat anti-F4/80 monoclonal 7 antibody conjugated with Alexa Fluor 488 (1:100) and goat anti-ChAT antibody (1:500) at 8 4°C. After washing twice with PBS for 2 min, the sections were incubated for 1 h with Alexa 9 Fluor 568 donkey anti-goat IgG (1:1000) at room temperature. 10 After the fluorescence staining, sections were incubated with Hoechst 33342 (1:500; 11 AnaSpec, Fremont, CA, USA) for 20 min at room temperature. After washing twice with 12 PBS for 2 min, the sections were mounted in VECTASHIELD Vibrance Antifade Mounting 13 Medium (Vector Laboratories, Newark, CA, USA) under a cover glass (Matsunami, Osaka, 14 Japan), and observed by confocal immunofluorescence microscopy (FV1000; Olympus, Tokyo, 15 Japan). 16 To determine whether the increase in subcutaneous WAT mass and whitening of 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 interscapular BAT were associated with lipid droplet hypertrophy, we performed 1 immunofluorescence analysis of WAT and BAT using rabbit anti-perilipin-1 antibody (1:400). 2 The acquisition of immunofluorescence images was performed using FV3000 (Olympus). 3 The nuclear density of adipocytes in WAT and BAT was determined by quantifying the 4 number of nuclei within perilipin-1 positive regions using ImageJ (https://imagej.net/ij/). 5 6 Oil Red O staining 7 8 To detect fat accumulation in the liver, hepatic tissues derived from WT and M4-KO mice 9 were fixed with 4% paraformaldehyde (Nacalai Tesque) for 3 h at 4°C, followed by washing 10 with PBS (pH 7.4). The tissues were subsequently embedded in Tissue-Tek OCT compound 11 (Sakura Finetek) and stored at -80°C before sectioning (10 µm) on a cryostat (CryoStar 12 NX70). Sections were air-dried for 1 h at 37°C and then rinsed with 60% isopropanol for 2 13 min at room temperature. The sections were then stained with Oil Red O solution 14 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 5 min at room temperature 15 and rinsed with 60% isopropanol for 2 min at room temperature. Next, the sections were 16 washed with tap water. Finally, the sections were mounted in VECTASHIELD Vibrance 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Antifade Mounting Medium (Vector Laboratories) under a cover glass (Matsunami) and 1 observed by inverted microscope (Axio Imager A2; ZEISS, Munich, Germany). 2 3 Quantitative analysis of the ACh content 4 5 Extracts for the determination of the ACh content were prepared from WT and M4-KO 6 subcutaneous adipose tissues. The tissues were ground to fine powder using TissueLyzer II 7 (Qiagen, Tokyo, Japan) under liquid nitrogen. The obtained powders were dissolved in 1 mL 8 of a 1:1 mixture of MilliQ water and acetonitrile (Nacalai Tesque), then vortexed. After 9 centrifugation at 200 × g for 1 min at 4°C, the supernatants were filtered twice in a spin 10 column filter (Millex-LH; Merck, Tokyo, Japan) at 90 × g for 1 min at 4°C, then the filtrates 11 were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). 12 The filtrates (10 μL) were applied to a LC-MS/MS system (LCMS-8060; Shimadzu) 13 equipped with an Intrada amino acid column (3.0 × 50 mm internal diameter, 3 μm; Imtakt, 14 Kyoto, Japan) operated at 40°C for chromatographic separation. The mobile phase consisted 15 of solvent A (0.2% (v/v) formic acid, 100 mmol/L ammonium formate, pH 4.0) and solvent B 16 (acetonitrile), and was delivered at a flow rate of 0.3 mL/min. The linear gradient used was 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 as follows: 0 to 5 min, 90% to 60% solvent B; 5 to 12 min, 60% solvent B; 12 to 15 min, 60% 1 to 30% solvent B; and 15 to 16 min, 30% to 90% solvent B. The electrospray ionization (ESI; 2 positive ionization mode) mass spectrometer was operated in multiple reaction monitoring 3 (MRM) mode to observe the transition of m/z 146.10 to m/z 87.05 for ACh quantification at 4 a collision energy of 15 (arbitrary unit). The retention time was 3.9 min for ACh. 5 6 Statistical analysis 7 8 Two-tailed unpaired Student's t tests were used to compare data between two groups. Data 9 and statistical analyses were performed using Microsoft Excel (Microsoft Corporation, 10 Redmond, WA, USA). Data are presented as the mean ± standard deviation (SD). In all cases, 11 differences were considered significant when p < 0.05. All experiments were repeated at 12 least three times. 13 14 15 16 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Results 1 2 mAChR-M4 deficiency caused weight gain with advancing age 3 4 It has been demonstrated that at around 12 weeks of age, mAChR-M4-deficient mice show 5 an increase in basal locomotor activity and are hypersensitive to the locomotor activity 6 stimulated through the D1 dopamine receptor (8). However, little is known about the impact 7 of the loss of function of mAChR-M4 with age. At 18 to 19 weeks of age, the body weight of 8 male M4-KO mice was significantly increased when compared to that of the control WT mice 9 (Fig. 1A). Notably, 20-week-old male M4-KO mice exhibited obesity while the age-matched 10 control mice did not (Fig. 1B). Also, the body weight of female M4-KO mice did not differ 11 from that of the control mice at 17 to 20 weeks of age (Fig. 1C and D). Subsequently, we 12 examined the body weight of the 20-week-old WT mice and mice deficient for each of the five 13 subtypes of mAChR (mAChR-M1 to mAChR-M5) in only the males. The body weight of the 14 mAChR-M4-deficient mice was significantly increased when compared to the WT mice (Fig. 15 1E). In contrast, body weight loss was observed in the mAChR-M3-deficient mice; this 16 decrease in body weight was thought to be due to dramatic hypoplasia of the anterior 17 AC CE PT ED M NU SC RI PT D ow nloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 pituitary gland associated with the greatly reduced pituitary growth hormone and prolactin 1 levels seen in our analyses of the brain of mAChR-M3-KO mice (20). Body weight loss was 2 also seen in the mAChR-M1-deficient and mAChR-M5-deficient mice, but the reason for the 3 weight loss remains unclear. In M4-KO mice, the weights of the epididymal adipose tissue, 4 subcutaneous adipose tissue, and mesentery adipose tissue were increased (Fig. 1F-J). Next, 5 we examined the food intake from 8 weeks to 20 weeks in the WT and M4-KO mice. Each 6 mouse was individually housed, and the weekly food intake was measured. The food intake 7 of the M4-KO mice was significantly higher at 9, 10, 11, 13, 16, and 18 weeks when compared 8 to that of the WT mice (Fig. 1K). However, almost no difference was seen in the average 9 weekly food intake over the entire study period between the WT and M4-KO mice (Fig. 1L). 10 As impaired energy expenditure in WAT is closely linked to the development of 11 obesity (21), we examined the physiological acclimation of M4-KO mice to nutrient overload. 12 When fed the standard chow diet, the energy expenditure in WAT differed between the M4-13 KO and WT mice (Fig. 1A and B). When fed the HFD, the M4-KO mice showed greater 14 weight gain than the WT mice even though the food intake was similar (Fig. 1M and N). 15 Based on these results, we hypothesized that mAChR-M4 signaling mediated by ACh may 16 provide essential protection against weight gain with advancing age and HFD-induced 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 obesity as well as other associated metabolic disorders, and that even partial loss of BAT 1 and/or beige fat regulation may have serious detrimental effects on systemic metabolism. 2 3 Deletion of Chrm4 induced lipid droplet hypertrophy in subcutaneous WAT, but 4 not in the liver 5 6 We next performed immunofluorescence analysis of subcutaneous WAT to determine 7 whether the increase in WAT mass was associated with lipid droplet hypertrophy. Anti-8 perilipin-1 antibody staining revealed that adipocyte in subcutaneous WAT of 20-week-old 9 male M4-KO mice was enlarged compared to that of WT male mice (Fig. 2A). Additionally, 10 we confirmed that nuclear density of adipocytes was significantly decreased in M4-KO mice 11 (Fig. 2B). The results revealed that the droplet in subcutaneous WAT was hypertrophic (Fig. 12 2A and B). As liver is the chief organ of lipid metabolism, we then examined the effect of 13 Chrm4 deletion on liver lipid accumulation. Oil Red O staining revealed that hepatic fat 14 content in M4-KO mice was not different from that in WT mice despite adiposity in the 15 subcutaneous WAT (Fig. 2C). Gene expression analysis was performed to determine whether 16 deletion of Chrm4 alters the gene expression pattern of lipid metabolism-related genes in 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 the liver. The qRT-PCR analysis revealed that the gene expression patterns for Fas and Scd 1 were significantly decreased, while the gene expression of Cd36 was significantly increased 2 (Fig. 2D). However, gene expression patterns for other genes were not significantly changed 3 (Fig. 2D). These data suggest that aberrant lipid metabolism in liver may not occur in M4-4 KO mice. 5 We found that mAChR-M4 deficiency caused weight gain with advancing age. 6 Accordingly, we examined to determine whether Chrm4 expressions decreased with 7 advancing age in WT mice. Chrm4 expression in subcutaneous WAT of 10-week- and 20-8 week-old male WT mice was examined with qRT-PCR. The result showed that Chrm4 9 expressions were significantly decreased accompanied by the aging process (Fig. 2E). 10 Additionally, we examined to determine whether there was difference between the 11 expression of Chrm4 in the subcutaneous WAT of male and female WT mice at 20 weeks of 12 age. The result showed that Chrm4 expression in subcutaneous WAT of male mice was 13 significantly decreased (Fig. 2F). These data indicate that sex-related decrease of Chrm4 14 expression solely occurs in male mice. 15 16 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Serum leptin levels in peripheral metabolism were increased in M4-KO mice 1 2 mAChR-M4 is expressed abundantly in the striatum and is also present at low levels in 3 several other brain regions, such as the cerebral cortex and hypothalamus (22-25). As M4-4 KO mice exhibited body weight gain and WAT accumulation, we hypothesized that 5 communication between the central nervous system, including the hypothalamus, and 6 peripheral metabolic tissues was impaired due to the lack of mAChR-M4 in the whole body. 7 To investigate the mediators of inter-organ communication, we monitored the blood glucose, 8 serum insulin, serum leptin, and plasma glucagon-like peptide 1 (GLP-1) levels in 20-week-9 old mice, because prolonged increases of these metabolites are known to be associated with 10 the development of obesity. The serum leptin levels significantly increased in the M4-KO 11 mice when compared to the control mice (Table 2). However, there was no significant 12 difference in the blood glucose (Fig. S1 (18)), serum insulin, and plasma GLP-1 levels 13 between the M4-KO and WT mice (Table 2). Additionally, we quantitatively measured the 14 neuropeptide Y (NPY) level in the brain using LC-MS/MS. NPY is involved in food intake, 15 obesity, and metabolic diseases (26). However, no significant difference was observed in the 16 NPY level in the brains of the M4-KO and WT mice (Table 2). 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 The brain, particularly the hypothalamus, regulates leptin signaling by sensing leptin 1 hormone, and by integrating and coordinating neurophysiological responses to control 2 whole-body metabolism. To investigate the underlying cause of the energy homeostasis in 3 the M4-KO mice, gene expression in the whole brain was examined by RNA-seq and qRT-4 PCR. There was no difference in the mRNA expression levels for genes related to energy 5 homeostasis, including for the genes encoding NPY, melanin-concentrating hormone (MCH), 6 and orexin, between the M4-KO and WT mice (Fig. 3A-D and Table S2 (18)). Interestingly, 7 in the brain of M4-KO mice, the expression level of the gene encoding NPGL was elevated, 8 but that of the gene encoding neurosecretory protein GM (NPGM) was not (Fig. 3E and F). 9 Both neurosecretory proteins have been shown to be essential for energy metabolism (27,28). 10 The increased expression of NPGL precursor mRNA in the brain of M4-KO mice may 11 contribute to the obese phenotype associated with elevated secretion levels of serum leptin 12 from WAT, which increases with WAT accumulation without any increase in dietary intake 13 (29). 14 15 16 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Deletion of Chrm4 increased subcutaneous WAT marker gene expression and 1 decreased BAT/beige marker gene expression 2 3 The body weight of 20-week-old male M4-KO mice was approximately 120% that of the 4 control mice (Fig. 1E); this increase in body weight appeared to be associated with an 5 increase in WAT (Fig. 1F-J). This indicated that mAChR-M4 directly targets WAT to control 6 the body weight. To examine the molecular mechanism, we performed RNA-seq using the 7 subcutaneous WAT of WT and M4-KO mice. The complete list of the sequenced genes is 8 provided in Supplementary Table S3 (18). As shown in Fig. 4A-C, the mRNA expression 9 levels of genes that are highly expressed in WAT, such as Fabp4, Lpl, and Lep, tended to be 10 increased in the subcutaneous WAT of M4-KO mice. We then carried out qRT-PCR to confirm 11 the expression levels of the genes, and found that they were significantly enhanced in the 12 subcutaneous WAT of M4-KO mice (Fig. 4F). In contrast, the expression levels of the genes 13 for adiponectin and resistin, which are commonly used as markers of WAT (30,31), tended 14 to be lower in the subcutaneous WAT of M4-KO mice at the transcript level (Fig. 4D and E). 15 Accordingly, we confirmed that the expression levels of the genes were significantly 16 decreased in the subcutaneous adipose tissue of M4-KO mice (Fig. 4F). Individuals with 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 obesity and type 2 diabetes have low plasma adiponectin levels (32). Resistin was first 1 discovered as an adipose-secreted hormone (adipokine) linked to obesity and insulin 2 resistance in mice (33). The level of WAT-derived resistin is increased in obese mice and is 3 strongly related to insulin resistance (34). However, it is possible that the serum level of 4 resistin does not correlate with the tissue mRNA levels (35). 5 Gene expression analysis was performed to determine whether deletion of Chrm4 6 alters the gene expression pattern of mRNAs encoding other mAChR subtypes in 7 subcutaneous adipose tissue. Loss of expression of Chrm4 was confirmed in the M4-KO 8 subcutaneous adipose tissue (Fig. 4G). The qRT-PCR analysis results revealed that in the 9 subcutaneous adipose tissue, the gene expression patterns for Chrm1, Chrm2, and Chrm5 10 were similar between the M4-KO and WT mice, while the gene expression of Chrm3 was 11 lower in the M4-KO mice than in the WT mice (Fig. 4G). mAChR-M3 deficiency resulted in 12 weight loss (Fig. 1E) (7). Decreased expression of Chrm3 may compensate for the body 13 weight gain of M4-KO mice. 14 The WAT accumulation in M4-KO mice suggested a decreased thermogenic capacity, 15 and thus, a decrease in energy expenditure in the subcutaneous WAT. Therefore, we 16 examined the expression pattern of BAT/beige adipose tissue marker genes. Deletion of 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Chrm4 tended to decrease the gene expression of the markers (Fig. 5A-H). In particular, a 1 statistically significant decrease was detected in the Car4 gene expression level (Fig. 5H). 2 We then carried out qRT-PCR analysis to confirm the expression levels of the genes. We 3 found that as the expression levels of WAT marker genes increased (with the exception of 4 the genes encoding adiponectin and resistin), the mRNA expression levels of the BAT/beige 5 adipose tissue-selective thermogenic genes, such as Tbx1, Ebf3, Ear2, Ucp1, Pdgfra, B3ar, 6 SP100, and Car4, progressively decreased (Fig. 5I). These data suggest the presence of 7 decreased BAT differentiation and/or impaired WAT and beige adipocyte differentiation in 8 the subcutaneous WAT of M4-KO mice. 9 10 Deletion of Chrm4 activated immune cells and the cell cycle, and impaired 11 adipogenesis 12 13 To gain a better understanding of the function of mAChR-M4 in subcutaneous WAT, 14 upregulated and downregulated DEGs were subjected to GO enrichment analysis. The GO 15 enrichment analysis results revealed that the upregulated DEGs were mainly enriched in 16 immune cell activation- and mitotic cell cycle-related processes, as illustrated in the GO 17 AC CE PT ED M NU SC RI PT D ow nloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 enrichment tree (Fig. 6A). In contrast, the downregulated DEGs showed no pattern of 1 categorization in the GO enrichment tree. 2 Obesity develops through an increase in adipocyte numbers through adipogenesis 3 and/or an increase in the size of the adipocytes. BAT plays a key role in thermogenesis, and 4 its activation to a state of increased energy expenditure is believed to protect against the 5 development of obesity. The 20-week-old male M4-KO mice exhibited obesity phenotypes 6 (Fig. 1B). We examined the shape and weight of the interscapular BAT, and the expression 7 patterns of WAT and BAT/beige adipose tissue marker genes in the M4-KO mice. The 8 interscapular BAT of the M4-KO mice had more profound whitening in part than that of the 9 WT mice (Fig. 6B). However, the size of the interscapular BAT was not significantly different 10 between the M4-KO and WT mice (Fig. 6C). We next performed an immunofluorescence 11 analysis of interscapular BAT to determine whether whitening of BAT is associated with 12 lipid droplet hypertrophy. Anti-perilipin-1 antibody staining revealed that there was no 13 difference between WT and M4-KO mice in interscapular BAT (Fig. 6D). Additionally, 14 nuclear density of the adipose cells was significantly decreased compared to that of WT mice, 15 suggesting that the droplet in interscapular BAT was not hypertrophic (Fig. 6D and E). We 16 observed a marked increase in WAT marker gene expression in the M4-KO mice (Fig. 6F). 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 The expression levels of all the BAT/beige adipose tissue marker genes (except for Car4) 1 were significantly increased in the interscapular BAT of the M4-KO mice when compared to 2 that of the WT mice (Fig. 6G). Uncoupling protein 1 regulates the thermogenic capacity of 3 adipocytes and contributes to the regulation of energy expenditure. These results suggest 4 that the WAT accumulation in the interscapular BAT of M4-KO mice may result from 5 impaired lipid metabolism with no decrease in Ucp1 expression. 6 7 mAChR-M4 localized to the subcutaneous WAT and AD-MSCs, and Chrm4 8 deletion had no impact on ChAT expression and the ACh content in 9 subcutaneous WAT 10 11 To identify the cells that were expressing Chrm4, we employed immunofluorescence 12 techniques to assess the cellular expression and tissue distribution of mAChR-M4. To attain 13 a high degree of sensitivity for detection, we used the mScarlet expression system in M4-KI 14 and M4-IRES-mScarlet reporter (M4-reporter) mice (Fig. 7A), which allowed detection of 15 the endogenous expression of mAChR-M4 from the expression of mScarlet. mAChR-M4 16 signals were observed in a small number of perilipin-positive adipose cells (Fig. 7B). 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Additionally, in triple-staining experiments carried out to see whether mAChR-M4 localized 1 to the AD-MSCs, mAChR-M4 signals were also observed in a small number of CD34 and 2 CD105-double positive cells (Fig. 7C). 3 We next attempted to identify the source of the ACh that acted on mAChR-M4 in 4 subcutaneous WAT. Double-staining experiments and confocal microscopy analysis results 5 revealed that ChAT-positive cells infiltrating the adipose parenchyma were positive for the 6 pan-macrophage cell surface marker F4/80, indicating that the double-positive cells were 7 resident interstitial macrophages (Fig. 7D). To further assess ChAT activity, we examined 8 the ChAT expression in the subcutaneous WAT of the WT and M4-KO mice. We found that 9 ChAT was expressed in the subcutaneous WAT of both the WT and M4-KO mice with no 10 significant difference (Fig. 7E). Next, we quantified the ACh content in the subcutaneous 11 WAT of the WT and M4-KO mice by LC-MS/MS. Results obtained from quantitative 12 measurements using a calibration curve demonstrated that the ACh content of the 13 subcutaneous WAT in the WT and M4-KO mice was 1.04 ± 0.05 nM and 1.15 ± 0.74 nM, 14 respectively, and there was no significant difference between the mice (Fig. 7F). Collectively, 15 the data showed that ACh signaling via mAChR-M4 occurs in the subcutaneous WAT of 16 mice. In healthy mice, the signal plays homeostatic functions that have yet to be identified. 17 AC CE PT D M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Discussion 1 2 Mice with whole-body KO of each of the five mAChRs have been created, and they showed 3 a variety of different phenotypes. It has been demonstrated that M4-KO mice (3 months of 4 age or older) show an increase in basal locomotor activity and are hypersensitive to the 5 stimulatory locomotor effect of D1 receptor activation (8). In peripheral tissues, M4-KO mice 6 present a striking hair phenotype, including retarded hair follicle morphogenesis and 7 insufficient follicular melanogenesis, when compared to age-matched WT controls (11). In 8 the present study, using 20-week-old M4-KO mice that exhibited obesity, we found an 9 additional function of mAChR-M4 in peripheral tissues: our results suggested that mAChR-10 M4 plays a role in mediating the accumulation of WAT, and thereby controls the body weight. 11 Obesity is a risk factor for other chronic diseases (36). Therefore, combating obesity 12 has been a major focus of health science. Our discovery that M4-KO mice gained weight with 13 age via the accumulation of WAT without any increase in food intake adds to the current 14 understanding of the etiology of obesity. Sex differences are known in several aspects of 15 obesity and the regulation of energy homeostasis in rodents and humans (37). We found that 16 20-week-old female M4-KO mice showed no weight gain. Further studies are needed to 17 AC CE PT ED AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 address the reason why the age-related weight gain did not occur in the female mice; it may 1 be related to female-specific events, including pregnancy. 2 In the current study, we also administered a HFD to M4-KO mice to see whether 3 they would show greater weight gain. As expected, compared to the control mice, the M4-4 KO mice gained weight relatively quickly when fed the HFD. Interestingly, experimental 5 evidence has indicated that several factors, including the consumption of a HFD, age, and 6 genetics, can induce the whitening of BAT and exacerbate obesity (38-40). Indeed, the 20-7 week-old M4-KO mice in the present study showed profound whitening, but not BAT 8 hypertrophy. Based on the results of our study, it appears that an increase in mAChR-M4 9 may increase the use of fat as an energy source. 10 An increasing number of neuropeptides and peptide hormones have been found to be 11 involved in energy homeostasis by regulating the dynamic equilibrium among energy intake, 12 storage, and expenditure (41). However, we observed no difference between the brains of WT 13 and M4-KO mice in regard to NPY, pro-MCH, and orexin gene expression. It has been 14 reported that cDNAs encoding the small secretory protein, NPGL were present in the 15 hypothalamus of birds and mammals (27,42,43). In addition, in transgenic mice generated 16 using the C57BL/6J strain to overexpress the NPGL gene (Npgl Tg mice), feeding of a 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 standard chow and high-calorie diets led to obesity (44). Additionally, the same research 1 group that identified NPGL also found a novel gene encoding NPGM, and NPGM-expressing 2 neurons were found to be key hypothalamic regulators of energy metabolism, in addition to 3 NPGL (28,42). We found that the mRNA expression level of NPGL was significantly 4 enhanced in the brain of M4-KO mice, while that of NPGM was not. Npgl Tg mice showed 5 fat accumulation without any increase in food intake, upregulation of the mRNA expression 6 levels for lipogenic factors in adipose tissue, or changes in blood parameters (29); these 7 findings are similar to those seen in the M4-KO mice, indicating that Chrm4- and NPGL-8 expressing neurons may work together to regulate energy metabolism. 9 Adipose tissues are classified based on their location, and physiological and 10 functional characteristics. We demonstrated that M4-KO mice show increased weight gain 11 with advancing age. In addition, the upregulation of marker genes specific to WAT was 12 detected in the subcutaneous WAT of M4-KO mice. Hyperleptinemia and leptin resistance 13 are often observed in obese mouse models (45). The manifestation of hyperleptinemia is 14 associated with age-related obesity. In healthy conditions, increased adiposity leads to more 15 leptin production. Increased levels of circulating leptin act on the hypothalamus and cause 16 metabolic shifts to regulate the energy balance (46,47). Previous studies have shown that 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 leptin resistance is caused by various mechanisms, including endoplasmic reticulum stress 1 and inflammation (48,49). The 20-week-old M4-KO mice were obese and had high serum 2 leptin levels. It is conceivable that the M4-KO mice may develop leptin resistance before the 3 onset of obesity. Leptin exerts its effect via two major neuronal populations, i.e., orexigenic 4 NPY/agouti-related protein neurons and anorexigenic pro-opiomelanocortin (POMC) 5 neurons, in the arcuate nucleus of the hypothalamus (50). Deletion of Chrm4 resulted in an 6 obese phenotype without any changes in the food intake. It has been shown that NPGL-7 immunoreactive fibers form close contacts with POMC neurons in the lateral part of the 8 arcuate nucleus, suggesting that NPGL may stimulate feeding behavior through the 9 inhibition of anorexigenic POMC neurons (27). In addition, Npgl overexpression in the ICR 10 mouse strain causes obesity with no increase in food intake (29), similar to that caused by 11 the deletion of Chrm4. Therefore, the coordination between Chrm4- and NPGL-expressing 12 neurons may mediate food intake and energy homeostasis via POMC neurons. Nevertheless, 13 it is unclear whether the M4-KO mice developed leptin resistance in the present study, and 14 further examinations are necessary. 15 BAT and beige adipose tissue are the opposite of WAT in that they promote energy 16 expenditure and counteract the complications linked to obesity. Our RNA-seq and qRT-PCR 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 analysis results showed that the expression levels of all marker genes for BAT/beige adipose 1 tissue were dramatically decreased, suggesting decreased energy expenditure. It is well-2 known that both BAT and beige adipose tissue are subject to a whitening effect that is 3 common in obesity; in the whitening effect, the tissues acquire unilocular fat cells that 4 gradually lose all the brown characteristics and gain WAT characteristics (51). Recent 5 research has demonstrated that BAT/beige adipose tissue whitening is a sophisticated 6 metabolic complication of obesity that is linked to multiple factors, such as diet, age, genetics, 7 thermoneutrality, and chemical exposure (52). Our data provide evidence that BAT 8 possesses a cell-intrinsic capacity to acquire a white-like state with advancing age. The 9 increased expression of Ucp1 may be a compensatory mechanism against certain stresses. 10 Interestingly, the upregulation of autophagy in the BAT of mice is consistent with the age-11 dependent decline of BAT activity and reduced metabolic rate (53). Future analyses of 12 autophagy during this transition are expected to uncover the fundamental mechanisms by 13 which mAChR-M4 controls BAT maintenance. 14 In rodents and humans, the mAChRs and nicotinic acetylcholine receptors expressed 15 in WAT are modulated by a variety of pathophysiological conditions, including the cold and 16 obesity (54-58). In the present study, we showed that mAChR-M4 is expressed in both 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 adipocytes and AD-MSCs in the subcutaneous WAT. AD-MSCs give rise to white, brown, or 1 beige adipose cells. The proper adipose tissue mass and function are essential for 2 maintaining metabolic health. As WAT lacks parasympathetic cholinergic innervation (59), 3 the ACh acting on adipocytes via ACh receptors cannot be of neuronal origin. It has 4 previously been reported that there are ACh-producing immune cells in WAT (58). Recently, 5 Severi and coworkers reported that ACh is synthesized and secreted by macrophages in 6 healthy mouse epididymal WAT (60). Our data also support the notion that macrophages 7 synthesize and secrete ACh. The secreted ACh can subsequently diffuse through the 8 extracellular space and affect the metabolism of adjacent adipocytes and AD-MSCs via 9 mAChR-M4 signaling. Although we have not yet investigated the possible effects of ACh on 10 adipocyte precursors, rodent AD-MSCs express mAChRs, and their activation affects 11 differentiation (61,62). It is possible that signaling through mAChR-M4 might inhibit white 12 adipose cell differentiation. 13 In conclusion, we demonstrated that the deletion of Chrm4 throughout the whole 14 body caused obesity with advancing age without any defects in energy homeostasis or 15 hyperphagia in mice. We also presented evidence that ACh signaling via mAChR-M4 may 16 have beneficial effects on white adipocytes. As ACh is a potent pleiotropic molecule, different 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 kinds of ACh receptors are likely found in all cell types. Better understanding of the 1 molecular mechanisms of the regulation of obesity by mAChR-M4 in WAT is expected to 2 provide novel approaches for conquering obesity with advancing age. However, more work 3 is needed to uncover the mechanism by which appetite- and energy homeostasis-related 4 genes, including NPGL, in the hypothalamus of M4-KO mice govern the risk of obesity with 5 advancing age. Targeting a specific population of neurons by using NPGL-Cre mice to 6 generate M4-KO mice would help clarify which neurons contribute to aberrant obesity due 7 to dysregulated gene expression caused by the deletion of Chrm4. 8 9 Acknowledgements 10 We thank Dr. Jürgen Wess (National Institute of Diabetes and Digestive and Kidney Diseases) 11 for providing the M1 to M5-/- mice. 12 13 Author contributions 14 TT designed and performed the experiments and wrote the manuscript. YT, AS, and SM 15 conducted the RNA-seq analysis. YT carried out the immunohistochemistry experiment. TW 16 carried out the quantitative ACh content analysis. SK carried out the breeding and 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 observation of mice. TY carried out the quantitative NPY content analysis. MS generated the 1 knock-out mice. All authors revised the manuscript. 2 3 Funding 4 This work was supported by Grants-in-Aid for Scientific Research (C) to TT (Grant numbers 5 JP20K06751 and JP23K05862). 6 7 Data Availability 8 All data generated and analyzed in this study are included in this article or in the data 9 repositories; Zendo repository for supplementary Figure S1 and supplementary Table 1-3 10 (18). 11 12 References 13 1. Hotamisligil GS. Inflammation and metabolic disorders. Nature. 2006;444:860-867. 14 2. Volpi E, Nazemi R, Fujita S. Muscle tissue changes with aging. Curr. Opin. Clin. Nutr. 15 Metab. Care. 2004;7:405-410. 16 3. Yoneshiro T, Aita S, Matsushita M, et al. Age-related decrease in cold-activated brown 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 adipose tissue and accumulation of body fat in healthy humans. Obesity (Silver Spring). 1 2011;19:1755-1760. 2 4. Cameron HL, Perdue MH. 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Piovesana R, Melfi S, Fiore M, Magnaghi V, Tata AM. M2 muscarinic receptor activation 15 inhibits cell proliferation and migration of rat adipose-mesenchymal stem cells. J. Cell 16 Physiol. 2018;233:5348-5360. 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 Figure legends 1 2 Figure 1. The body weight and WAT weight in aging M4-/- mice. (A) and (B) Changes in the 3 body weight in male mice. *P < 0.05 for WT versus M4-/- mice. (C) and (D) Changes in the 4 body weight in female mice. (E) Changes in the body weight in male mice at the age of 20 5 weeks. **P < 0.01, ***P < 0.001 for WT versus M1-/-, M3-/-, M4-/-, or M5-/- mice. (F) and (G) 6 Epididymal adipose tissue in male M4-/- mice at the age of 20 weeks. (H) and (I) 7 Subcutaneous adipose tissue in male M4-/- mice at the age of 20 weeks. (J) Mesentery 8 adipose tissue in male M4-/- mice at the age of 20 weeks. (K) Weekly food intake of male M4-9 /- mice from 8 weeks to 20 weeks of age. (L) Average weekly food intake of male M4-/- mice 10 over the experimental period. (M) and (N) Body weight of WT and M4-/- mice during the 11 feeding of a high-fat diet (HFD). All data are expressed as the mean ± SD. Data were 12 analyzed using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. 13 14 Figure 2. Morphological and mRNA expression analyses in the subcutaneous adipose tissue 15 and the liver of WT and M4-KO mice. (A) Anti-perilipin-1 antibody staining of subcutaneous 16 adipose tissue. (B) Analysis of nuclear density of the subcutaneous adipose cells in WT and 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 M4-KO mice. (C) Oil Red O staining of the liver sections. (D) Relative quantification of the 1 expression level of lipid metabolism-related genes in the liver of M4-KO mice. (E) Relative 2 quantification of the expression level of Chrm4 in the subcutaneous adipose tissues of 10-3 weeks- and 20-weeks-old male WT mice. (F) Relative quantification of the expression level 4 of Chrm4 in the subcutaneous adipose tissues of male and female WT mice at 20 weeks of 5 age. The results of qRT-PCR are based on three independent experiments and are presented 6 as the mean ± SD. Data were analyzed using a two-tailed Student’s t-test. **P < 0.01, ***P 7 < 0.001. 8 9 Figure 3. Comparison of the expression of marker genes in the brain of WT and M4-KO mice. 10 (A) to (C) Differential expression patterns of Npy, Pmch (Pro-Mch), and Hcrt (orexin) in the 11 brain of M4-KO mice. The results of RNA-seq are based on three independent experiments 12 and are presented as the mean ± SD. (D) Relative quantification of the expression level of 13 Npy, Mch, and Hcrt in the brain of M4-KO mice. (E) and (F) Relative quantification of the 14 expression level of NPGM and NPGL in the brain of M4-KO mice. The results of qRT-PCR 15 are based on three independent experiments and are presented as the mean ± SD. Data 16 were analyzed using a two-tailed Student’s t-test. *P < 0.05. 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 1 Figure 4. Comparison of the expression of WAT marker genes in the subcutaneous adipose 2 tissue of WT and M4-KO mice. (A) to (E) Differential expression patterns of Fabp4, Lpl, Lep, 3 Adiponectin, and Resistin in the subcutaneous adipose tissue of M4-KO mice. The results of 4 RNA-seq are based on three independent experiments and are presented as the mean ± SD. 5 (F) Relative quantification of the expression level of Fabp4, Lpl, Leptin, Adiponectin, and 6 Resistin in the subcutaneous adipose tissue of M4-KO mice. (G) Relative quantification of 7 the expression level of muscarinic receptor genes in the subcutaneous adipose tissue of M4-8 KO mice. The results of qRT-PCR are based on three independent experiments and are 9 presented as the mean ± SD. Data were analyzed using a two-tailed Student’s t-test. **P < 10 0.01, ***P < 0.001. 11 12 Figure 5. Comparison of the expression of BAT/beige adipose tissue marker genes in the 13 subcutaneous adipose tissue of WT and M4-KO mice. (A) to (H) Differential expression 14 patterns of Tbx1, Ebf3, Ear2/Nr2F6, Ucp1, Pdgfa, B3ar/Adrb3R, Sp100, and Car4 in the 15 subcutaneous adipose tissue of M4-KO mice. The results of RNA-seq are based on three 16 independent experiments and are presented as the mean ± SD. Data were analyzed using a 17 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 two-tailed Student’s t-test. *P < 0.05. (I) Relative quantification of the expression level of 1 Tbx1, Ebf3, Ear2/Nr2F6, Ucp1, Pdgfa, B3ar/Adrb3R, Sp100, and Car4 in the subcutaneous 2 adipose tissue of M4-KO mice. The results of qRT-PCR are based on three independent 3 experiments and are presented as the mean ± SD. Data were analyzed using a two-tailed 4 Student’s t-test. **P < 0.01, ***P < 0.001. 5 6 Figure 6. Gene set enrichment analysis of the subcutaneous adipose tissue of M4-KO mice, 7 and comparison of the expression of WAT and BAT/beige adipose tissue marker genes in the 8 BAT of WT and M4-KO mice. (A) GO enrichment tree shows that genes related to immune 9 cell activation and cell division were upregulated in the subcutaneous adipose tissue of M4-10 KO mice. The enrichment P-value is indicated for each functional category. (B) and (C) The 11 BAT in male M4-KO mice at the age of 20 weeks. (D) Anti-perilipin-1 antibody staining of 12 brown adipose tissue. (E) Analysis of nuclear density of the brown adipose cells in WT and 13 M4-KO mice. (F) Relative quantification of the expression level of Fabp4, Lpl, and Leptin in 14 the BAT of M4-KO mice. (G) Relative quantification of the expression level of Tbx1, Ebf3, 15 Ear2/Nr2F6, Ucp1, Pdgfa, B3ar/Adrb3R, Sp100, and Car4 in the BAT of M4-KO mice. The 16 results of qRT-PCR are based on three independent experiments and are presented as the 17 AC CE PT ED M NU SC RI PT D ow nloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025 mean ± SD. Data were analyzed using a two-tailed Student’s t-test. **P < 0.01, ***P < 0.001. 1 2 Figure 7. Localization of mAChR-M4 and ChAT in M4-reporter and M4-KI mice, and 3 comparison of the expression of ChAT and the ACh content in the subcutaneous adipose 4 tissue of WT and M4-KO mice. (A) Structures of transgenes integrated into the genomic 5 DNA of M4-KI and M4-reporter mice. IRES, internal ribosome entry site. (B) Double 6 staining using antibodies with red fluorescent protein (RFP; red) and perilipin (green). (C) 7 Triple staining using antibodies with RFP (red), CD34 (green), and CD105 (gray). (D) Double 8 staining using antibodies with RFP (red) and F4/80 (green). Nuclei were stained using 9 Hoechst staining (blue). White scale bars represent 20 μm. (E) Relative quantification of the 10 expression level of Chat in the subcutaneous adipose tissue of M4-KO mice. (F) The ACh 11 content of the subcutaneous adipose tissue of M4-KO mice was measured quantitatively by 12 LC-MS/MS analysis. The results of qRT-PCR and LC-MS/MS are based on three 13 independent experiments and are presented as the mean ± SD. Data were analyzed using a 14 two-tailed Student’s t-test. 15 16 AC CE PT ED M AN US CR IP T Downloaded from https://academ ic.oup.com /endo/advance-article/doi/10.1210/endocr/bqaf064/8105668 by guest on 07 April 2025
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